Amplification of nucleic acids is widely used in research, forensics, medicine and agriculture. Polymerase chain reaction (PCR) is the most widely used method for in vitro DNA amplification. A PCR reaction typically utilizes two oligonucleotide primers that are hybridized to the 5′ and 3′ borders of the target nucleic acid molecule and a DNA polymerase that extends the annealed primers by polymerizing deoxyribonucleotide-triphosphates (dNTPs) to generate double-stranded products. By cycling the temperature of the reaction mixture (known as thermocycling), the two strands of the DNA product are separated and can serve as templates for the next round of annealing and extension, and the process is repeated, resulting in exponential amplification of the template DNA.
Other nucleic acid amplification methods have also been developed that do not rely on thermocycling. These methods are broadly categorized as isothermal amplification methods due to the fact that they do not rely on repeated cycles of temperature change to operate. However, current isothermal amplification methods require multiple enzymes, lack the specificity of PCR reactions or require four or more primers targeting six or more regions of the template nucleic acid for efficient amplification. A new isothermal amplification technique would be useful that combines simple design (e.g., at most four primers targeting at most five template regions), cost-efficiency (e.g., using a single enzyme), and robustness (e.g., amplification at temperatures ≧60° C.) for the development of, for example, field and point of care testing.